Overexpression or mutation of the 1-synuclein protein causes familial Parkinson's Disease (PD). Recent studies in yeasts and nematodes have suggested that the primary cause of 1-synuclein toxicity is a deficit in the endoplasmic reticulum (ER)-to-Golgi transport step of the secretory pathway. Our lab has documented the first evidence that 1- synuclein is in fact a potent inhibitor of mammalian ER-to-Golgi transport. Furthermore, our experiments indicate that 1-synuclein acts directly to inhibit ER-to-Golgi machinery prior to the onset of cellular stress responses or accumulation of large protein aggregates. I propose to use this fellowship support to elucidate the precise mechanism by which 1-synuclein does this with the following specific aims: (1) Identify the precise step(s) in ER-to-Golgi transport inhibited by 1-synuclein. A series of high resolution in vitro assays that reconstitute key subreactions in ER- to-Golgi transport will be employed. (2) Determine the specific molecular interactions between 1-synuclein and the transport machinery that disrupt ER-to-Golgi transport. We will elucidate the in vitro and in vivo binding interactions between 1-synuclein and the ER-to-Golgi transport machinery and determine how this binding affects the machineries' macromolecular structures and activities. (3) Extend findings to dopaminergic neuroendocrine cells. I will validate my findings using dopaminergic neuroendocrine cells-a cellular model for the cells killed in PD. Our lab has already developed and fully established the required in vitro assays, which have been used productively for years to study the ER/Golgi transport machinery. I now hope to have the opportunity, at this watershed moment in PD research, to apply our unique expertise and technology to elucidate exactly how 1-synuclein perturbs this fundamental subcellular system, ultimately leading to neurodegeneration and PD. PUBLIC HEALTH RELEVANCE: Overexpression or mutation of the 1-synuclein protein in humans leads to the selective death of dopamine-producing neurons, the manifested pathology of PD. New therapies to mitigate PD would require research to pinpoint the harmful action(s) of the 1-synuclein protein on a specific subcellular pathway(s). This research will identify how 1- synuclein affects subcellular transport mechanisms, providing new and unanticipated avenues for therapeutic intervention or prevention.